Antagonist of ligands and uses thereof

ABSTRACT

The invention provides multivalent ligand binging agents (traps) for members of the TGF-β superfamily and polypeptide linkers and methods for making and using such constructs. In an embodiment of the invention there is provided a multivalent binding agent with affinity for a member of the TGF-β superfamily, said agent comprising the general structure I: (&lt;bd1&gt;-linker1) k -[{bd1&gt;-linker2-&lt;bd2&gt;-linker3 f -} n -(&lt;bd3&gt;) m -(linker4-&lt;bd4&gt;) d ] h , where: -n and h are independently greater than or equal to 1; -d, f, m and k arc independently equal to or greater than zero; -bd1, bd2, bd3 and bd4 are polypeptide binding domains having an affinity for the same member of the TGF-β superfamily, with bd1, bd2, bd3, and bd4 being independently the same or different from each other; and, -linkeri, linker2, linker3 and linker4 are unstructured polypeptide sequences; wherein the number of amino acids in each linker is determined independently and is greater than or equal to X/2.5; where, X equals the shortest linear distance between: (a) the C-terminus of an isolated form of the binding domain that is located at the N-terminus of the linker and that is specifically bound to its ligand; and, (b) the N-terminus of an isolated form of the binding domain that is located at the C-terminus of the linker and that is specifically bound to its ligand.

This application claims priority from U.S. Provisional Application No.60/907,059 filed Mar. 19, 2007

FIELD OF INVENTION

The invention relates to the field of antagonists and, morespecifically, to polypeptide antagonists capable of use as single chainmultivalent ligand traps.

BACKGROUND OF INVENTION

Many undesirable biological processes occur via ligand binding to cellsurface receptors. Thus, it is sometimes desirable to have compounds andmethods to reduce or modulate such binding.

The TGF-β superfamily includes a number of ligands of biologicalsignificance.

TGF-β and Activin play critical pathogenic roles in many diseasesincluding the progression of cancer and uncontrolled fibrosis andscarring of tissues, e.g. kidney, lung and liver fibrotic diseases.Furthermore, Myostatin/GDF8 is another ligand which is related toActivin and which shares binding to the same Type II receptor(ActivinRIIb). Myostatin is a powerful inhibitor of skeletal musclegrowth and is a validated therapeutic target for muscle wasting diseasessuch as muscular dystrophy. Bone morphogenetic proteins (BMP), which areother ligands in the TGF-β family, have been implicated incardiovascular diseases. For example, high levels of both BMP2 and BMP4have been found in calcified atherosclerotic plaques and diseased aorticvalves.

Principal agents that target these ligands are ligand traps/antagoniststhat bind and sequester ligand. Two examples are: 1) anti-ligandantibodies and 2) soluble receptor ectodomains.

Efforts have been made to identify methods to reduce ligand binding bytrapping ligand and preventing its interaction with the cell surfacereceptors. Inhibition of certain ligands has been reported usinganti-ligand antibodies that trap and neutralize the ligand directly. Fortherapeutic and diagnostic applications, however, antibodies areproblematic, particularly due to issues arising from theirimmunogenicity (and the danger of adverse immune response in patients)and their large size (restricting their ability to reach targets outsidethe bloodstream).

Soluble versions of receptor ectodomains antagonize ligands directly bybinding to them and preventing them from interacting with cell surfacereceptors. In the case of TGF-β, in animal models, expression of a TGF-βreceptor type II (TβRII) ectodomain (ED) partially restored hostimmunity and promoted tumor clearance, indicating that receptorectodomain—mediated neutralization of TGF-β inhibits tumor progression.It has been shown, however, that the efficacy of monovalent TβRII toantagonize TGF-β is less than could be desired. Attempts to overcomethis led to the production of an artificially dimerized form of versionsof TβRII-ED, dimerized, via fusion to either coiled-coil domains or theFc domain of IgG. This dimerization improved the antagonist effect.

Bivalent receptor-based traps/neutralizers that antagonize multimericligand activity have the potential to act as therapeutic or diagnostic(imaging or non-imaging) agents for diseases/disorders caused byover-production/activity of the target ligand. It has been demonstratedthat non-covalent dimerization of TβRII-ED (for example, via fusion toheterodimerizing coil strands (coiled-coil TβRII-ED)), greatly enhancesthe antagonist potency of TβRII-ED (De Crescenzo et al., 2004, J. Biol.Chem. 279: 26013). A significant disadvantage of the coiled-coil fuseddimer is that the non-covalent nature of the dimerization domain limitsits potency, i.e. it dissociates at low concentrations such that a largeportion of the coil-fused receptor ectodomain will be acting as amonomer rather than a dimer. Use of the Fc domain of IgG provides acovalent interaction, but at the cost of large size and increasedprobability of immunogenicity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. Depicts embodiments of amino-acid sequences corresponding tointrinsically unstructured regions in the extracellular portions ofselect TGF-β-superfamily receptors.

FIG. 1B. Depicts embodiments of amino-acid sequences corresponding tostructured ligand-binding domain regions in the extracellular portionsof select TGF-β-superfamily receptors.

FIG. 2A. Depicts examples of embodiments of in-line fused receptorectodomains as homo-bivalent single-chain traps of several TGF-β-familygrowth factors. The “l” sign indicates the point of fusion.

FIG. 2B. Depicts examples of sequences corresponding to natural linkersof embodiments of homo-bivalent single-chain traps resulting from fusionof the entire extracellular portions of select TGF-β-superfamilyreceptors.

FIG. 2C. Depicts examples of sequences corresponding to embodiments ofartificial linkers for homo-bivalent single-chain traps at varyingsequence identity to natural linker sequences.

FIG. 2D. Depicts examples of sequences corresponding to varying thelinker length for embodiments of homo-bivalent single-chain traps bydeleting or repeating of natural sequences, or by inserting ofartificial sequences, into the natural linker sequence.

FIG. 3. Depicts an illustration of an embodiment of the (TβR-II)²single-chain trap construct on a three-dimensional molecular mechanicalmodel of the (TβR-II)² single-chain trap bound to the TGF-β growthfactor. Two 90°-rotated views are provided.

FIG. 4. Depicts diagrams relating to the feasibility of specificembodiments of trap constructs with natural linkers fromthree-dimensional structural models. Shown are molecular mechanicsenergy-minimized natural linkers for embodiments of (TbR-II)2,(ActR-IIb)2 and (BMPR-Ia)2 homo-bivalent single-chain traps in complexwith the TGF-β3, Activin and BMP-2 growth factors, respectively. Eachgrowth factor covalent dimer is rendered in gray. Each single-chain trapis rendered in black, and consists of two folded binding domains and inintervening unstructured linker. Each dot indicates the point of fusionin the linker region between two receptor ectodomains to generate thesingle-chain trap. Arrowheads indicate polypeptide chain direction inthe trap's linker. Two 90°-rotated views are provided for each complex.

FIG. 5A. Depicts molecular dynamics (MD) model for an embodiment of the(TβR-II)2 homo-bivalent single-chain trap bound to the TGF-β3 growthfactor (right images). An initial model with energy-minimized linker andwith ligand-binding domains in crystallographic positions bound onto thegrowth factor is also shown for reference (left images, see also FIGS. 3and 4). The single-chain trap is rendered in black and the growth factorcovalent dimer is rendered in gray. Ten time-averaged structures (eachover 1 ns) covering 10 ns timeframe of MD simulation are overlaid. Two90°-rotated views are provided

FIG. 5B. is a graphical representation of per residue root-mean-square(RMS) fluctuations of an embodiment of the (TβRII)^(2 l /TGF-β)3complex, time-averaged over the last 10 ns of MD simulation.

FIG. 5C. is a graphical representation of solvated Interaction Energy(SIE) between an embodiment of a single-chain (TβRII)² trap and theTGF-β3 ligand over the last 10 ns of MD simulation of their complex,with an average value of −25.4 kcal/mol.

FIG. 6. Depicts a schematic of embodiments of prototype (TβRII)² andmodified N-His (TβRII)² traps.

FIG. 7A. Depicts surface plasmon resonance (SPR)-based biosensor(Biacore™) sensograms showing an embodiment of a prototype (TβRII)² (indiluted conditioned media from different % transfections) binding tosurface-immobilized TGF-β3 ligand.

FIG. 7B. Depicts surface plasmon resonance sensograms comparing bindingof embodiments of bivalent prototype (TβRII)², bivalent TβRII-Fc andmonovalent TβRII to 270 RUs surface-immobilized TGF-β3 ligand.

FIG. 8. Is a photographic depiction of a gel showing high levelproduction and purification yield of an embodiment of N-His (TβRII)²protein from 500 ml culture of transfected 293 cells.

FIG. 9A. Is a graphical depiction of inhibition of TGF-β signaling inMv1Lu luciferase reporter cells by an embodiment of prototype (TβRII)²compared to TβRII-Fc.

FIG. 9B. Is a graphical depiction of SPR-based determination of trapbinding of TGF-β in solution by an embodiment of prototype (TβRII)² andTβRII-Fc compared to monomeric TβRII-ED.

FIG. 9C. Is a graphical depiction of inhibition of TGFβ1-induced 4T1cell invasion in vitro by an embodiment of prototype (TβRII)² andTβRII-Fc traps.

FIG. 10A. Is a Biacore™ sensogram showing direct binding of embodimentsof N-His (TβRII)² and monomeric N-His TβRII to different isoforms ofTGF-β.

FIG. 10B. Is a graphical depiction of a Biacore™ comparison ofperformance of embodiments of 100 nM N-His (TβRII)² and TβRII-Fc to bindto 500 RUs each of TGF-β1 or TGF-β3

FIG. 10C. Is a graphical depiction of SPR-based determination of IC50for trap binding to TGF-β1 (5 nM) in solution. The graph shows efficientbinding of TGF-β1 by an embodiment of a N-His (TβRII)² trap and TβRII-Fctrap versus reduced binding by monomeric TβRII (293 cell-produced or E.coli-produced).

FIG. 10D. Is a graphical depiction showing efficient inhibition of TGF-βsignaling in Mv1Lu luciferase reporter cells by an embodiment of N-His(TβRII)² and TβRII-Fc compared to poor inhibition by monomeric TβRII(293 cell-produced and E-Coli-produced).

FIG. 11. (A) is a photographic depiction and (B) is a graphicaldepiction of results showing that an embodiment of N-His (TβRII)²exhibits long-term stability and activity in 10% serum at 37° C.

FIG. 12. Provides graphical depictions showing efficient neutralizationof TGF-β1 (A) and binding of TGF-β1 in solution (B) by an embodiment ofa (TβRII)² trap (ligand binding agent) having a 60 amino acid linker.

FIG. 13. Is a graphical depiction showing efficient inhibition ofMyostatin signaling in A204 cells by an embodiment of an (ActRIIB)² trap(ligand binding agent) compared to the less potent inhibition ofActRIIB-Fc and monomeric ActRIIB.

FIG. 14. Is a graphical depiction of results showing that an embodimentof a bivalent (BMPR1a)² trap (ligand binding agent) is more potent thanmonovalent BMPR1a trap for neutralization of BMP2.

FIG. 15A. Provides schematic diagrams exemplifying embodiments ofin-line fusions of receptor ectodomains leading to embodiments ofheterovalent single-chain traps of TGF-β-superfamily growth factors.

FIG. 15B parts 1 and 2. Depict embodiments of amino-acid sequencesexemplifying embodiments of heterovalent single-chain traps (ligandbinding agents) of TGF-β-superfamily growth factors, and correspondingto the domain organization diagrams depicted in FIG. 15A.

SUMMARY OF INVENTION

The invention relates to ligand binding agents capable of permittingmodulation of cellular response to members of the TGF-β superfamily bybinding one or more members of the TGF-β superfamily and preventinginteraction with cellular receptors, and methods of designing and usingsuch agents. The ligand binding agents taught herein are preferablysingle chain multivalent ligand binding agents. However, it would bepossible to link such single-chain constructs to other uni- ormultivalent molecules and/or to combine two or more such single chaintraps using multimerization domains known in the art (e.g. coiled-coildomains, Fc domains, pentabodies) to form a multimeric trap if sodesired and any such trap having a multivalent single chain portionfalls within the scope of the present invention.

In an embodiment of the invention there is provided methods andprocesses to engineer multivalent receptor ectodomains using asingle-chain approach.

The ligand binding agents of the invention are preferably multivalentligand traps, having at least two binding domains (bd) which recognizedifferent sites on (or the same site of different portions of) the samemember of the TGF-β superfamily. The binding domains may be modified,for example to facilitate purification, so long as such modifications donot reduce binding affinity to unacceptable levels.

The binding domains (bd) of the ligand traps are preferably joined by aflexible polypeptide linker region. This linker should preferablyinclude an unstructured amino acid sequence which in some embodiments iseither the same as or derived from conservative modifications to thesequence of a natural unstructured region in the extracellular portionof the receptor for the ligand of interest or another receptor in theTGF-β superfamily. In other instances, such linkers may be entirelyartificial in composition and origin but will contain amino acidsselected to provide an unstructured flexible linker with a lowlikelihood of encountering electrostatic or steric hindrancecomplications when brought into close proximity to the ligand ofinterest.

In some instances, the linker will include regions to facilitatepurification (e.g. His tags) or to facilitate the addition of cargo oraccessory molecules. When such additions affect the unstructured natureof the linker or introduce potential electrostatic or steric concerns,appropriate increases to the linker length will be made to ensure thatthe two binding domains are able to bind their respective sites on theligand. In light of the methods and teachings herein, suchdeterminations could be made routinely by one skilled in the art.

In an embodiment of the invention there are provided ligand traps havingthe general Structure I:(<bd1>-linker1)_(k)-[{<bd1>-linker2-<bd2>-linker3_(f)-}_(n)-(<bd3>)_(m)-(linker4-<bd4>)_(d)]_(h),where:

-n and h are independently greater than or equal to 1;

-d, f, m and k are independently equal to or greater than zero;

-bd1, bd2, bd3 and bd4 are polypeptide binding domains having anaffinity for the same member of the TGF-β superfamily, with bd1, bd2,bd3, and bd4 being independently the same or different from each other;and,

-linker1, linker2, linker3 and linker4 are unstructured polypeptidesequences;

wherein the number of amino acids in each linker is determinedindependently and is greater than or equal to X/2.5; where,

X equals the shortest linear distance between:

(a) the C-terminus of an isolated form of the binding domain that islocated at the N-terminus of the linker and that is specifically boundto its ligand; and,

(b) the N-terminus of an isolated form of the binding domain that islocated at the C-terminus of the linker and that is specifically boundto its ligand.

As used herein “an isolated form” of a binding domain is a form of thatbinding domain acting as a monovalent monomer.

Subject to the constraints described herein, linkers 1, 2, 3, and 4 maybe the same or different. In certain embodiments the linker is between25 and 60 amino acids in length Also provided are nucleic acid sequencesencoding such ligand traps.

Depending on the values selected for d, f, h, k, m, and n, the ligandtrap structure may comprise a large number of repeating units in variouscombinations or may be a relatively simple structure such as StructureII <bd1>-linker-<bd2>.

In certain embodiments of the invention, the member of the TGF-βsuperfamily to which the binding domains (bd) have affinity is selectedfrom the group consisting of: TGF-β1, TGF-β2, TGF-β3, activin βA,activin βB, activin βC, activin βE, bone morphogenic protein (BMP) 2,BMP 3, BMP4, BMP 5, BMP 6, BMP 7, BMP 8, BMP 9, BMP 10, BMP 11, BMP 12,BMP 13, BMP 14, BMP 15, growth differentiation factor (GDF) 1, GDF 3,GDF 8, GDF 9, GDF 15, Nodal, Inhibin α, anti-Mullerian Hormone, Lefty 1,Lefty 2, arteman, Persephin and Neurturin.

In an embodiment of the invention there is provided a binding agentwherein one or more of bd1, bd2, bd3, and bd4 is selected from one ofSEQ ID NO 43-48.

In an embodiment of the invention the binding agent comprises one ormore of SEQ ID No 75 to 82.

In an embodiment of the invention the binding agent comprises one ormore of SEQ ID NO 31-42 or 49-74 as a linker sequence.

The invention also provides a method of designing a multivalent bindingagent useful in modulating responsiveness of a cell to a member of theTGF-β superfamily, said method comprising:

a) identifying a member of the TGF-β superfamily of interest;

b) obtaining two polypeptide binding domains having affinity fordifferent sites on the member of the TGF-β superfamily member;

c) obtaining an unstructured polypeptide linker of at least a number ofamino acids equal to (X/2.5) where

X equals the shortest linear distance between:

-   -   (i) the C-terminus of an isolated form of the binding domain        that is located at the N-terminus of the linker and that is        specifically bound to its ligand; and,    -   (ii) the N-terminus of an isolated form of the binding domain        that is located at the C-terminus of the linker and that is        specifically bound to its ligand; and,

d) modelling the linker between the binding domains and carrying outmolecular dynamics simulations to substantially minimize molecularmechanics energy and reduce steric and electrostatic incompatibilitybetween the linker and the member of the TGF-β superfamily.

The design method can optionally be expanded to further include a stepe) of producing a fusion protein comprising the two polypeptide bindingdomains joined by the unstructured polypeptide linker.

The ligand binding agents disclosed herein are also useful inpurification of ligand, for example, by immobilization on an inertmatrix on a solid support, on, for example, to nanoparticles toconcentrate levels of ligand in a sample.

The invention also provides novel polypeptide sequences useful in avariety of applications. These sequences include SEQ ID NOs 53 to 74.Also provided are nucleic acid sequences encoding these polypeptidesequences.

Also provided is a method of modulating the response of a cell to TGF-βin its environment, said method comprising exposing the cell to amultivalent ligand trap comprising a ligand binding agent (ligand trap)disclosed herein.

In an embodiment of the invention there is provided a binding agenthaving the general structure V:

Wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, may be the same ordifferent, may not be present and when present, may independently be oneor more of a fusion protein for targeting, a single domain antibody, aradiotherapy agent, an imaging agent, a fluourescent dye, a fluorescentprotein tag, a cytotoxic agent for chemotherapy a nano particle-basedcarrier, a polymer-conjugated to drug, nanocarrier or imaging agent, astabilizing agent, a drug a nanocarrier and a dendrimer and a supportfor use in purification or concentration of ligand; and wherein bd1,bd2, bd3, bd4, linker1, Iinker2, Iinker3, linker4, k, f, n, m, d, and hare defined as in Structure I. In light of the disclosure herein, oneskilled in the art can select suitable R-groups for diagnostictherapeutic or other applications.

In an embodiment of the invention there is provided an isolatedpolypeptide having at least 80%, 85%, 90%, 95%, 98%, 99% and 100%sequence identity to a natural unstructured region in the extracellularportion of a receptor for a member of the TGF-β superfamily and beingsubstantially free of structured regions capable of specific binding toa member of the TGF-β superfamily. In some instances, this isolatedpolypeptide has at least 80%, 85%, 90%, 95%, 98% , 99% sequence identityto one or more of SEQ ID NO 31-42 and SEQ ID NOs 49-74.

In an embodiment of the invention there is provided a polypeptidecomprising a region having at least 80%, 85%, 90%, 95%, 98%, 99%sequence identity to one or more of SEQ ID NOs 53-74 and SEQ ID NOs82-118. In some instances this polypeptide has a region with at least90%, 95%, 98%, 99% sequence identity to one or more of SEQ ID NOs 53-74.

In an embodiment of the invention there is provided a polypeptide havingbetween 43% and 99% sequence identity to a naturally unstructured regionin the ectodomain of a receptor for a member of the TGF-β superfamily.

In an embodiment of the invention there is provided a nucleic acidsequence encoding a polypeptide disclosed herein.

In an embodiment of the invention there is provided a method ofmodulating the response of a cell to a TGF-β superfamily member in itsenvironment, said method comprising exposing the cell to a ligandbinding agent disclosed herein.

In an embodiment of the invention there is provided a data storagemedium comprising instructions for determining the minimum linker lengthwhen designing a ligand binding agent.

In an embodiment of the invention there is provided a data storagemedium comprising a means for identifying acceptable minimal linkerlength when designing a ligand binding agent.

Linker length will be considered acceptable when it permits binding ofbinding domains located on each of the N- and C-termini of the linker tobind their natural binding sites on their natural ligand such that, withboth binding domains so bound, the ligand is bound with a higheraffinity than it would be bound by binding of only one of the bindingdomains.

BRIEF DESCRIPTION OF THE INVENTION

In an embodiment of the invention there is provided a single-chainnon-naturally occurring polypeptide useful as a ligand binding agent.The ligand binding agent comprises structured ligand-binding domains(denoted bd) derived from or based on the extracellular portion of anatural receptor or receptors, joined by one or more polypeptidelinkers. The ligand binding agent provides a multivalent binding agentand does not require fusion to any conventional dimerizing ormultimerizing moieties such as coiled-coil domains of Fc domains inorder to be multivalent.

In an embodiment of the invention, there is provided a multivalentbinding agent with affinity for a member of the TGF-β superfamily, saidagent comprising the general structure I:

(<bd1>-linker1)_(k)-[{<bd1>-linker2-<bd2>-linker3_(f)-}_(n)-(<bd3>)_(m)-(linker4-<bd4>)_(d)]_(h),

where:

-n and h are independently greater than or equal to 1;

-d, f, m and k are independently equal to or greater than zero;

bd1, bd2, bd3 and bd4 are polypeptide binding domains having an affinityfor the same member of the TGF-β superfamily, with bd1, bd2, bd3, andbd4 being independently the same or different from each other; and,

-linker1, linker2, linker3 and linker4 are unstructured polypeptidesequences;

wherein the number of amino acids in each linker is determinedindependently and is greater than or equal to X/2.5; where,

X equals the shortest linear distance between:

(a) the C-terminus of an isolated form of the binding domain that islocated at the N-terminus of the linker and that is specifically boundto its ligand; and,

(b) the N-terminus of an isolated form of the binding domain that islocated at the C-terminus of the linker and that is specifically boundto its ligand.

As used herein “an isolated form” of a binding domain is a form of thatbinding domain acting as a monovalent monomer.

The length of the linker is considered to be the number of amino acidsbetween:

(a) the C-terminal main chain carbon atom of the binding domain locatedat the linker's N-terminal end; and

(b) the N-terminal main-chain nitrogen atom of binding domain located atthe linker's C-terminal end.

Non-limiting examples of useful linkers are found in the amino acidsequences in SEQ ID NOs 53 to 74 which should be read conventionallywith the N-terminus on the left and the C-terminus on the right, and incorresponding reverse sequences having the same amino acids but whereinthe C-terminus is on the left and the N-terminus is on the right as thesequences are written in full. In some embodiments, such reversesequences will preferably be produced using D-amino acids. Whereimmunogencity is of concern, it will generally be desired to screen suchreverse sequences for immunogenicity at an early stage. (For examples ofreverse sequences, see SEQ ID NOs 82-118). All amino acids sequences inthis document are written N-terminus to C-terminus unless otherwisenoted. All sequences disclosed herein except SEQ ID NOs 82-118 aredisclosed as using L-amino acids and the use of a D-amino acid isconsidered a variant affecting the percent sequence identity to thesequences as stated.

In an embodiment of the invention, the ligand binding agent has thegeneral Structure II:

<bd1>-linker2-<bd2>.

In an embodiment of the invention, the ligand binding agent has thegeneral Structure III

<bd1>-(linker2-<bd2>)_(n).

In an embodiment of the invention, the polypeptide has the generalStructure IV:

([bd1]-[linker1]-bd1)_(f)-[linker2]-([bd2]-[linker3]-[bd3])_(g),

when f and g are greater than or equal to one.

In an embodiment where bd2 and bd3 are the same, and f and g are thesame number, this can result in a substantially mirror symmetricstructure around linker 2, subject to differences in the linkers. Ininstances where bd3 is different from bd2 and and/or where f and g aredifferent numbers, different structures will be produced. It is withinthe capacity of one of ordinary skill in the art to select suitablebinding domains, linkers, and repeat frequencies in light of thedisclosure herein.

In some instances, the binding domain region of the single-chainpolypeptide will be selected for its ability to bind a growth-factorligand having a covalently-stabilized dimeric quaternary structure, andmay be selected from a list of growth factors from within the TGF-βfamily, e.g., transforming growth factor beta (TGF-β, bone morphogeneticprotein (BMP), activin, myostatin, and including their naturallyoccurring isoforms.

In some instances, the polypeptide is designed to bind simultaneously toequivalent but spatially distinct sites on a multimeric ligand. As usedherein “multimeric” includes dimeric, trimeric, and greater numbers ofunits, and “multivalent” includes bivalent, trivalent, and greaternumbers of binding domains.

In some instances, the linker is independently selected to have varyingdegrees of sequence identity to naturally occurring unstructured aminoacid sequences found in the native receptor sequence in the regionsflanking the ligand binding domain, for example 70%, 80%, 90%, 95%, 98%,99% or 100% sequence identity, whereas for entirely artificial linkers(e.g. poly-Gly or poly-Ser linkers, sequence identity will be evenlower. Examples of linker sequences of varying degree of identity to thenatural receptor sequence are given in FIG. 2C. and SEQ ID NOs 82-118(Table II).

In some instances, the number of amino acid residues in the linker ofeither natural or artificial origin is selected to be equal to orgreater than the minimum required distance for simultaneous (bridged)binding to two binding sites on the growth factor to be bound by therelevant binding domains. An example of an embodiment of such adetermination is given in the section “Feasibility assessment procedurefor designed single-chain bivalent traps”. Examples of natural andartificial linker sequences of varying length are given in FIG. 2D andSEQ ID NOs 82-118. In some instances, linker length is between 18-80a.a., 25-60 a.a., 35-45 a.a.

In some instances, the overall molecular mass of bivalent agentsdisclosed herein before glycosylation is between about 29 kDa and 37kDa, and the overall mass following typical glycosylation is betweenabout 40 kDa and 60 kDa. Thus, there is provided herein, multivalentligand traps having a pre-glycosylation size of between about 12 kDa and19 kDa per binding domain.

The ligand traps disclosed herein will generally have a lower molecularmass than comparable multimeric ligand traps constructed using knownmultimerization domains.

Example of Selected Ligand Trap Sizes

Actual (with glycosylation) Agent Predicted for protein based onSDS-PAGE (TβRII)² 34 kDa 50-60 kDa (TβRIIb)² 37 kDa 50-60 kDa (ActRIIB)²30 kDa 50-60 kDa (BMPR1a)² 29 kDa 40-50 kDa RIIEcoil + RIIKcoil 37 Kd +40 kDa = 77 kDa TβRII-Fc 60 Kd + 60 kDa = 120 kDa

Polypeptides of the invention can be useful as therapeutic agents thatneutralize the action of disease-associated covalently-stabilizeddimeric ligands such as growth factors. They may also have commercialpotential for use as diagnostic agents to detect the presence ofdisease-associated covalently-stabilized dimeric ligands such as growthfactors in imaging and non-imaging diagnostic applications. They canalso be useful in the purification and/or concentration or segregationof ligand in vitro.

DETAILED DESCRIPTION OF INVENTION

Although the invention is described with reference to specific examples,it will be understood that it is not so limited.

Experiment #1: Design Strategy of Single-Chain Bivalent Traps forTGF-β-Family Ligands

1. Single-chain recombinant traps were designed against growth factorsthat belong to the transforming growth factor TGF-β superfamily ofcysteine-knot cytokines according to SCOP (Andreeva et al., 2008, Nucl.Acid Res. 36: D419) and Pfam (Finn et al., 2006, Nucl Acid Res. 34:D247) structural classifications. More specifically, these growthfactors including, for example, TGF-βs, activins and BMPs, share thesame 3D architecture and form covalent disulfide-linked homodimers. Themethod disclosed herein is applicable to all members of the TGF-βsuperfamily, including TGF-β1, β2, -β3; activin βB, βC, βE; bonemorphogenetic proteins (BMP) 2-15; growth differentiation factors (GDF)1, 3, 8 (myostatin), 9 and 15; Nodal; Inhibin α; anti-Mullerian hormone(AMH); Lefty 1 and 2; Arteman, Persephin and Neurturin.

2. Single-chain recombinant traps against TGF-β superfamilygrowth-factors were designed from the extracellular portion of theircognate natural receptors. The extracellular segment of all these TGF-βsuperfamily receptors contain a single structured domain that belongs tothe snake-toxin family according to SCOP (Andreeva et al., 2008, Nucl.Acid Res. 36: D419) and Pfam (Finn et al., 2006, Nucl Acid Res. 34:D247) structural classifications. The complete extracellular portion ofthese receptors typically includes unstructured segments flanking theirfolded ligand-binding domain. These unstructured extracellular portionswere apparent from the experimentally determined 3D structures availablefrom the PDB database (Berman et al., 2000, Nucl. Acid Res. 28: 235),e.g., crystal structures for type II TGF-β receptor ectodomain (Hart etal., 2002 Nat. Struct. Biol. 9: 203; Boesen et al., 2002, Structure 10:913; Groppe et al., 2008, Mol. Cell 29: 157), type I TGF-β receptorectodomain (Groppe et al., 2008, Mol. Cell 29:157), type IIa activinreceptor ectodomain (Allendorph et al., 2006, Proc. Natl. Acad. Sci. USA103: 7643), type IIb activin receptor ectodomain (Thompson et al., 2003,EMBO J. 22: 1555; Greenwald et al., 2004, Mol. Cell 15: 485), type I BMPreceptor ectodomain (Kirsch et al., 2000, Nat. Struct. Biol. 7: 492), orthe NMR structure of the type II TGF-β receptor ectodomain (Deep et al.,2003, Biochemistry 42: 10126)]. In the absence of experimental data, asfor example in the case the extracellular region of the IIb splicingvariant of the TGF-β type II receptor, unstructured extracellularsegments were defined by: (i) sequence portions falling outside of thefolded ligand-binding domain boundaries located by comparative analysisagainst structurally characterized homologs, and (ii) predictions basedon knowledge-based algorithms, e.g., DISOPRED (Ward et al., 2004, J.Mol. Biol. 337: 635). Amino acid sequences corresponding to theunstructured (i.e., flexible) and structured (i.e., folded,ligand-binding domain) regions from the ectodomains of several receptorsof TGF-β-superfamily growth factors, are given in FIGS. 1A and 1B,respectively..

3. Homo-bivalent single-chain recombinant traps hereby designed againstTGF-β-superfamily growth factors disclosed herein were designed withregard to the experimentally determined binding mode betweenTGF-β-family ligands and the extracellular portion of their cognatenatural receptors. The ligand-receptor binding mode was provided atatomic level by the high-resolution 3D structures available for severalmembers of the TGF-β-superfamily ligands in complex with their cognatereceptor ectodomains. Examples of experimental molecular structures forTGF-β-superfamily-growth-factor/receptor ectodomain complexes includeTGF-β3 bound to TβR-II-ED (Hart et al., 2002 Nat. Struct. Biol. 9: 203),activin bound to ActR-IIb-ED (Thompson et al., 2003, EMBO J. 22: 1555;Greenwald et al., 2004, Mol. Cell 15: 485), BMP-2 bound to type Ia BMPreceptor ectodomain BMPR-Ia-ED (Kirsch et al., 2000, Nat. Struct. Biol.7: 492) and ActR-IIa-ED (Allendorph et al., 2006, Proc. Natl. Acad. Sci.USA 103: 7643), BMP-7 bound to ActR-IIa-ED (Greenwald et al., 2003, Mol.Cell 11: 605). These structures provided the relative spatialorientation between two separate receptor ectodomain chains (molecules)binding simultaneously onto one covalently homodimerized ligandmolecule, i.e., 2:1 receptor:ligand stoichiometry. Higher-orderligand-receptor assemblies between a particular TGF-β-superfamily growthfactor and ectodomains from different receptor types have also beendetermined, for example the ternary complexes between TGF-β3, TβR-II-EDand TβR-I-ED (Groppe et al., 2008, Mol. Cell 29:157) or between BMP-2,ActR-IIa-ED and BMPR-Ia-ED (Allendorph et al., 2006, Proc. Natl. Acad.Sci. USA 103: 7643). These structures provide the relative spatialorientation between four separate receptor ectodomain chains (molecules)binding simultaneously onto one covalently homodimerized ligandmolecule, i.e., 2:2:1high-affinity-receptor:low-affinity-receptor:ligand stoichiometry. Suchstructures were used as guides to design hetero-bivalent,hetero-trivalent and hetero-tetravalent single-chain traps ofTGF-β-superfamily growth factors and are useful in designingsingle-chain traps for other suitable ligands of interest involving theTGF-(3 superfamily.

4. Homo-bivalent single-chain traps of TGF-B-family ligands weretherefore designed as unnatural fusion proteins consisting of thesequence (excluding the signal peptide) of the natural extracellularportion of the receptor repeated twice. FIG. 2A presents schematicallyhomo-bivalent single-chain traps with natural linkers for threeTGF-B-family ligands, where structured and unstructured regions arebased on experimental data for single-domain extracellular portions, aspresented in FIGS. 1A and 1B. This resulted in constructs with twostructured domains for binding to select TGF-β-superfamily ligand(s),spaced by an unstructured flexible linker formed by fusing theunstructured C-terminus of the first domain to the unstructuredN-terminus of the second domain. The natural linker was alsoprogressively substituted by artificial sequences as well as varied inlength (FIGS. 2B-D). From thermodynamic and kinetic considerations, itwas expected that divalent receptor ectodomains would provide increasedligand-binding affinities and slower ligand-dissociation rates relativeto single- domain receptor ectodomains.

Experiment #2: Feasibility Assessment Procedure for DesignedSingle-Chain Bivalent Traps

To the extent to which the structures of various TGF-β-superfamilygrowth factors are conserved, the structures of their cognate receptorectodomains are conserved, and the 2:1 receptor-ligand bindingstoichiometry is conserved, the concept of fusing two natural receptorectodomain sequences to produce single-chain homo-bivalent traps withimproved in vitro ligand binding affinity and cellular ligandneutralizing activity relative to respective monovalent receptorectodomains, is applicable to the entire family of TGF-β family. Thefeasibility of these ligand traps can be theoretically assessedroutinely by following the stepwise procedure outlined below. Althoughthe procedure is presented for homo-bivalent single-chain traps, it alsoapplies to other designs covered here, e.g., hetero-bivalent andhetero-tetravalent single-chain traps.

1. The linear distance is measured between the C-terminal main-chaincarbon atom of one domain and the N-terminal main-chain nitrogen atom ofthe other domain when bound to the covalently-dimerized ligand.Alternate structures of the complex reflecting internal geometricalflexibility in the homodimerization mode of the disulfide-stabilizedligand when bound to the receptor ectodomains, as reported in severalcases (Greenwald et al., 2004, Mol. Cell 15: 485), can be included inthe design process. A computer hardware equipped with commercial/publicsoftware appropriate for manipulating molecular structures on anavailable graphics device can be routinely employed to this end.

2. The linear distance (in Å units, 1 Å=10⁻¹⁰ m) is divided by a factorof 2.5 to calculate the minimum number of amino acid residues that theflexible linker should posses (Table 1) in order to allow simultaneousbinding of the folded domains to their binding sites on the homodimericligand. The 2.5 factor is based on the Cα-Cαextent of fully extendedlinkers, which peaks at 3.0 Å (George and Heringa, 2002, Protein Eng.15: 871), minus an average tolerance of 0.5 Å per amino acid residue toallow for deviations of the linker path from linearity.

(Table 1. Linker characteristics for select examples of single-chaintraps of TGF-β-family growth factors. Minimum number of residuesrequired for linkage represents the structure-based linear distance forlinkage (Å) divided by a factor of 2.5.)

3. The number of amino acid residues in the unstructured linker portionof the bivalent single-chain trap should be at least equal to theestimated minimum number of linker residues required. Receptor isoformsthat differ in the length of the extracellular unstructured segments,such as the TGF-β receptor isoforms II and IIb (FIG. 2B), can beincluded in the design process. The natural sequence-based linker canalso be shortened up to the estimated minimum number of amino acidresidues without significantly impairing the ligand binding affinity andneutralizing activity of the trap. A preferable location for shorteningthe unstructured linker is from the point of fusion (see FIG. 3) ineither or both directions relative to the amino acid sequence. Exampleof shortened natural linkers that can be utilized in single-chain trapdesign are given in FIG. 2D. As listed in Table 1, the required minimallength of the linker varies between various single-chain traps ofTGF-β-superfamily growth factors. An upper limit for the length of theunstructured linker is not defined. Hence, ligand binding agent (trap)constructs with linkers comprising unstructured sequence segmentsrepeated in whole or in part are envisioned to comply with bivalentdesign and preserve the desired characteristics of the trap. The naturallinker can be progressively substituted by artificial sequences, whichmay or may not result in different linker lengths. Examples of linkerslonger than the natural linker designed by repeating of natural sequenceor by introducing of artificial sequence are given in FIG. 2D.

4. Finally, atomic-level theoretical analysis is to be carried out,where the linker is modeled between the structured domains and themolecular structure of the trap-ligand complex is refined by minimizingthe molecular mechanics energy and by carrying out molecular dynamicssimulations (Cornell et al., 1995, J. Am. Chem. Soc. 117: 5179). Thismay, in some cases, highlight regions of steric and/or electrostaticincompatibility between the trap's linker and the growth-factor, andsuggest that the length and/or composition of the linker may beincompatible with the bivalent design, even if the linker complies withthe minimum number of amino acids requirement as per step (3.) above. Ifthe linker can be accommodated without affecting the simultaneousbinding of the structured domains to their binding sites on the ligand,then the trap construct is deemed feasible for the proposed application.Computer hardware equipped with commercial/public software appropriatefor manipulating molecular structures on an available graphics device,and for performing energy calculation and simulation based on molecularmechanics force fields, e.g., the AMBER force field (Cornell et al.,1995, J. Am. Chem. Soc. 117: 5179), can be routinely employed by oneskilled in the art in order to carry out this structural modelinganalysis. A detailed molecular modeling analysis of the (TβR-II)²homo-bivalent single-chain trap is provided as an example in thefollowing section and includes molecular dynamics simulation. Examplesof molecular mechanics energy-refined models of three single-chainhomo-bivalent traps: (TβR-II)², (ActR-IIb)² and (BMPR-Ia)², bound totheir respective growth factors are shown in FIGS. 3 and 4. Theseatomic-level models represent starting points for further computer-basedoptimization of linker composition and length.

This process is explained in greater detail in the example below:

Modeling Example A, Experiment #2

i. In one example, the atomic-level solution structure of thesingle-chain homo-bivalent trap (TβR-II)² was simulated in complex withthe growth factor TGF-β3.

The starting point for molecular design of the (TβR-II)² trap was the2.15 Å-resolution crystal structure of the disulfide-linked dimerichuman TGF-β3 complexed with two TGF-β type II receptor ectodomains (Hartet al., 2002 Nat. Struct. Biol. 9: 203), deposited in the Protein DataBank (Berman et al., 2000, Nucl. Acid Res. 28: 235) under the code 1KTZ.Because this structure displays the growth factor in a non-canonicalconformation probably due to the low pH used in the crystallizationconditions (Hart et al., 2002 Nat. Struct. Biol. 9: 203; Groppe et al.,2008, Mol. Cell 29:157), the binary complex structure was firstreconstructed with the ligand in canonical conformation as reportedpreviously (Hinck et al., 1996, Biochemistry 35: 8517; Mittl et al.,1996, Protein Sci. 5: 1261), which was also recently confirmed by theternary structure of TGF-β ligand-receptor assembly (Groppe et al.,2008, Mol. Cell 29:157). An initial 3D molecular model of the (TβR-II)²trap incorporating an inter-domain natural linker of 35 amino-acidresidues (as per sequence listed in FIG. 2B) was constructed fromstandard geometries followed by conjugate-gradient energy minimizationof the molecular mechanics force field energy using an AMBER all-atomforce field (Cornell et al., 1995, J. Am. Chem. Soc. 117: 5179) and theAMBER 9 suite of programs (Case et al., 2005, J Comput. Chem. 26: 1668).During energy minimizations, only the linker regions of the traps wereallowed to move, while the coordinates of the growth factors and of thefolded domains of the traps were fixed. The resulting 3D molecular modelof the homo-bivalent single-chain trap (TβRII)² bound to TGF-β3 isdepicted in FIG. 4.

This initial model of the complex was used as input for moleculardynamics (MD) simulation carried out together with AMBER FF03 forcefield (Duan et al., 2003, J. Comput. Chem. 24: 1999; Lee & Duan, 2004,Proteins 55: 620) within the AMBER 9 suite of programs (Case et al.,2005, J. Comput. Chem. 26: 1668). The molecular system consisting of 245amino-acid residues from the single-chain trap (from the full-length(TβR-II)² trap with 272 amino-acid residues, 21 unstructured residuesfrom the N-terminus and 6 flexible residues from the C-terminus were notincluded in the MD simulation), 224 amino-acid residues of the TGF-β3dimer and 14 Na⁺ counterions (added to maintain electroneutrality) wassolvated in rectangular water box using the

Xleap program in the AMBER 9 software. The distance between the wall ofthe box and the closest atom of the solute was 12.0 Å, and the closestdistance between the solute and solvent atoms was 0.8 Å. The entiresystem was energy-minimized by applying harmonic restraints with forceconstants of 10 kcal/mol/Å² to all solute atoms, followed by heatingfrom 100K to 300K over 25 ps in the canonical ensemble (NVT), and byequilibrating to adjust the solvent density under 1 atm pressure over 25ps in the isothermal-isobaric ensemble (NPT) simulation. The harmonicrestraints were then gradually reduced to zero with four rounds of 25 psNPT simulations. After additional 25 ps simulation, a 15 ns productionrun was obtained with snapshots collected every 1 ps. For allsimulations, 2 fs time-step and 9 Å non-bonded cutoff were used. TheParticle Mesh Ewald method (Darden et al., 1993, J. Chem. Phys. 98:10089) was used to treat long-range electrostatics, and bond lengthsinvolving bonds to hydrogen atoms were constrained by SHAKE (Ryckaert etal. 1977, J. Compt. Phys. 23: 327). No other constraints were imposedduring the MD simulation.

As seen from FIG. 5A, the single-chain trap (TβR-II)² bound to TGF-β3attains a stable MD solution structure that preserves the simultaneousbinding of the two ligand-binding domains onto the dimeric growth factoras observed in the crystal structure of unlinked receptor ectodomains(Hart et al., 2002 Nat. Struct. Biol. 9: 203, Groppe et al., 2008, Mol.Cell 29:157). This substantiates the feasibility of the designedsingle-chain TGF-β trap in terms of the length of the linker. The MDanalysis also reveals that in the complex, the linker region of thesingle-chain trap becomes relatively rigid, with only 6 residuesexperiencing greater mobility (expressed as per-residue andtime-averaged root-mean-square fluctuations) than the rest of the trap'samino acid residues (FIG. 5B). In addition, the single-chain trapestablished favorable interaction with the growth factor, as evaluatedby the solvated interaction energy function for scoring protein-ligandbinding affinity (SIE) (Naim et al., 2007, J. Chem. Inf. Model. 47:122). A highly favorable SIE value of −25.4 kcal/mol was calculated asan average over the last 10 ns of MD simulation (FIG. 5C). This furtherindicates the feasibility of the employed natural linker in terms ofamino acid composition, that is, there were no significant unfavorablesteric and electrostatic contacts predicted between the trap's linkerand the growth factor.

ii. In one example, structure-based design leads to a divalent moleculeconsisting of two human T6RII ectodomains that are fused in tandem intoa single polypeptide chain (schematically shown in FIG. 6). In thisconstruct, an intervening linker sequence is formed from theunstructured natural C-terminal sequence of one ectodomain (black, 10residues) and the unstructured natural N-terminal sequence of the otherectodomain (white, 25 residues). This linker bridges between the twostructured TGF-β-binding domains. This TGF-β trap is hereby namedprototype (TβRII)². The construct also contains an N-terminal myc tagand C-terminal 6×His tag for ease of detection and protein purification.In the prototype (TβRII)² the native IPP sequence is replaced by GGRwithin the linker due to a NotI restriction site inserted duringconstruction of the (TβRII)² gene. Also shown is another construct witha 35 amino acid residues linker with native IPP restored, and having aN-terminal His tag. This construct is termed “modified N-His” (TβRII)²and features a native linker sequence. Predicted molecular models of(TβRII)² bound to TGF-β are given FIGS. 3-5.

Experiment #3: Small Scale Production of Prototype (TβRII)² andDemonstration of TGF-β-Binding Activity

FIG. 6 shows a schematic of prototype (TβRII)². The prototype (TβRII)²gene was cloned into mammalian expression vector pTT and increasingamounts were transiently transfected into HEK293 cells. The conditionedmedia from these transfected cells were collected after 5 days andtested via SPR Biacore analysis for the binding of secreted (TβRII)² toa TGF-β3 surface (FIG. 7A). The sensogram shows increasing levels ofbinding that correlates with cells transfected with increasing levels of(TβRII)² plasmid (ranging from 1% to 95% transfected cells), indicatinga dosage effect and specific binding. The binding characteristics of(TβRII)² (produced from 95%-transfected cells) was compared withdimerized TβRII-Fc and monomeric TβRII (FIG. 7B). The sensogram ofprototype (TβRII)² was similar to the TβRII-Fc interaction (slow offrate), and both were distinct from monomeric TβRII interaction (fast offrate), indicating that (TβRII)² interacts with the TGF-β3 surface in ahigh-affinity, bivalent manner.

Experiment #4: Production and Purification of Prototype and ModifiedN-His (TβRII)²

Scaleup production of prototype (TβRII)² in 293 cells resulted invariable yields of protein (1-3 mgs per 1 liter culture) uponpurification via cobalt column, perhaps due to a less accessible His tagat the C-terminus. A modified version was constructed having aN-terminal His tag, termed N-His (TβRII)², as shown in FIG. 6. N-His(TβRII)²-transfected HEK293 cells were grown in 500 ml culture. Themedia was collected, concentrated 5-fold by 10 kDa Centricon filtrationand then passed through a 10 ml Fractogel Cobalt column. FIG. 8 shows aSDS-PAGE analysis of N-His (TβRII)² at the various stages ofpurification. The N-His (TβRII)² in the eluted fractions (lane 6) isrelatively pure and migrates as a smear (likely due to glycosylation) inthe 50-60 kDa range. The total yield from 500 ml culture was 7-8 mgs,indicating that the N-His (TβRII)² protein is amenable to large-scaleproduction.

Experiment #5: Demonstration that (TβRII)² is a Potent TGF-β Trap

The ability of purified prototype (TβRII)² to neutralize TGF-β wastested on Mv1Lu cells having a TGF-β-responsive luciferase reporter geneand compared with TβRII-Fc from two sources, commercial R&D andcollaborator H. Lin (FIG. 9A). The resulting inhibition curves indicatedthe average IC₅₀ for prototype (TβRII)² is 0.58 nM (S.D. 0.64) which isin the same range as for TβRII-Fc Lin (0.45 nM) and slightly higher thanTβRII-Fc R&D (0.1 nM). Purified prototype (TβRII)² was also comparedwith dimeric TβRII-Fc and monomeric TβRII-ED for their ability tocompetitively bind TGF-β in solution via Biacore analysis (FIG. 9B).Increasing amounts of each binder was added separately to a constantamount of TGF-β1 or -β3 (5 nM) followed by coinjection of this mixtureover a TGF-β-specific antibody surface. The level of unbound TGF-β atequilibrium is assessed by the maximum/plateau level of the surfacebinding curve (FIG. 9B) Prototype (TβRII)² and TβRII-Fc have similarlylow IC50s in the range of 5-8 nM, as would be expected forintra-molecular, divalent binding of TGF-β. In contrast, the IC50 formonovalent TβRII-ED is 10-20 fold higher. One might predict, for fullavidity, that the IC50 for dimeric (TβRII)² could be at least 100-foldgreater than for monomeric TβRII-ED. In order to augment avidity,variable linker lengths may be sampled for (TβRII)² (see FIGS. 2C and2D). These results (FIGS. 9A and 9B) indicate that (TβRII)² is anexcellent trapping/neutralizing reagent for TGF-β and hence is a goodcandidate therapeutic and/or diagnostic agent for diseases in whichTGF-β is causative and overexpressed/overactive (e.g. breast tumors). Tothis end we examined the ability of prototype (TβRII)² to preventTGF-β-induced invasion of 4T1 breast cancer cells in vitro (FIG. 9C).Similar to TβRII-Fc, prototype (TβRII)² reduced 4T1 cell invasion toapproximately 20% of the non-trap treated (+TGF-β) control.

Experiment #6: Assessment of Binding Characteristics and Efficacy ofN-His (TβRII)²

The two different SPR Biacore assays that were utilized in FIG. 7 andFIG. 9B were used again to characterize the N-His (TβRII)²-TGF-β ligandinteraction. First, the direct binding assay was utilized where theTGF-β trap was injected over various immobilized TGF-β isoform surfaces.While this assay can verify binding to different TGF-β isoform surfaces,it cannot verify that a 1:1 trap: TGF-β homodimer interaction isoccurring in solution due to the nature of using an immobilized TGF-βsurface. In order to show trap binding enhancement to soluble TGF-βligand, indirect binding assays were carried out in which a constantTGF-β concentration was preincubated with various trap or TβRII monomerconcentrations and then injected over a 1D11 antibody surface(anti-TGF-β1 to 3). In this manner, the 1D11 surface measures the amountof free (or unbound) TGF-β. A lower IC50 indicates binding enhancementdue solely to avidity. In the direct binding assay, the binding ofbivalent N-His (TβRII)² to immobilized TGF-β1 and 3 was compared to thatof the monomeric N-His TβRII construct (FIG. 10A). N-His (TβRII)² boundto all TGF-β isoforms (1-3), showing a fast on rate and significantlyslower off rate of binding to TGF-β1 and β3 compared with monomericN-His TβRII, as is expected for a bivalent binding interaction. Inaddition, N-His (TβRII)² showed binding to TGF-β2 whereas monomericTβRII binding to this isoform was undetectable. We also compared bindingof N-His (TβRII)² and TβRII-Fe to TGF-β1 and β3 (FIG. 10B). Both trapsshowed similar binding kinetics with characteristic fast on rates andslow off rates. In order to assess trap binding to ligand in solution,the indirect binding assay to determine IC50s as carried out using 5 nMTGF-β1. IC50 curves for bivalent N-His (TβRII)² , TβRII-Fc andmonovalent TβRII (produced either in 293 cells or E. coli) weregenerated (FIG. 10C). N-His (TβRII)² and TβRII-Fc both showed efficientbinding, having IC50s of 1.1 and 1.6 nM, respectively. The IC50s ofN-His TβRII (293 cells) and TβRIIED (E. coli) were approximately 8 and70 fold higher (respectively) than that of N-His (TβRII)². Similardifferences between bivalent and monovalent traps were observed inneutralization assays using Mv1Lu luciferase reporter cells (FIG. 10D).The IC50s for N-His (TβRII)² and TβRII-Fc in this assay were in the subnM range whereas monomeric N-His TβRII (293 cells) showed only partialneutralization in the 10-100 nM range, and monomeric TβRII (E. coli) wasunable to neutralize TGF-β. The results also show that, compared toprototype (TβRII)², the modified N-His (TβRII)² was most efficient inneutralizing TGF-β (compare FIGS. 9A and 10D).

Experiment #7: N-His (TβRII)² Exhibits Long-Term Stability and Activity

The susceptibility of the N-His (TβRII)² to proteolytic degradation wasassessed by incubating N-His (TβRII)² in the presence of 10% fetalbovine serum at 37° C. for a period of 7 days (FIG. 11). The westernblot on the left shows that N-His (TβRII)² protein remains intactthroughout the 7 day period. In addition, the neutralization curves onthe right demonstrate that N-His (TβRII)² retains its activity. Theseresults show that N-His (TβRII)² is not adversely sensitive toproteolysis and therefore is a good candidate therapeutic and/or imagingagent for animal studies.

Experiment #8: The N-His (TβRIIb)², which has a Long Linker (60 AminoAcids, see FIGS. 1A and 2A) is more Potent than N-His (TβRII)²

The IC50 for N-His (TβRIIb)² for neutralization of TGFβ1 was 0.04 nM(FIG. 12A), which is 4-fold more potent than N-His (TβRII)² (IC50=0.16nM, FIG. 10D). Similarly, when tested by SPR analysis (Biacore) forbinding TGF-β1 in solution, N-His (TβRIIb)² was more potent that N-His(TβRII)² (Fig.12B). These results illustrate that modification of linkerlength is at least one parameter whereby trap efficiency can beimproved.

Experiment #9: (ActRIIb)²: another Example of a Single-Chain ReceptorTrap within the TGF-β Family

In order to show that the single-chain bivalent receptor strategy taughtherein can be applied to other ligands of the TGF-β family, (ActRIIb)²(shown schematically in FIG. 2A) was constructed from the human ActRIIbreceptor using this strategy. ActRIIb is the high affinity receptor forboth myostatin and activin B. (ActRIIb)² and monomeric ActRIIb wereproduced in 293 cells and their ability to neutralize myostatin wastested using human rhabdosarcoma A204 cells. These cells have theActRIIb receptor and were transfected with (CAGA)₁₂-luciferase reportergene (responsive to activin and myostatin) (FIG. 13). (ActRIIb)²exceeded the neutralization potency of monomeric ActRIIb (IC50 of 0.1and 0.38 nM, respectively), thus demonstrating the better bindingefficiency of this bivalent trap. In addition, (ActRIIb)² was 10-foldmore potent than dimeric ActRIIb-Fc. These results therefore indicatethat the single-chain receptor strategy taught herein can be used as aplatform technology to develop effective trapping reagents of otherligands within the TGF-I3 family.

Experiment #10: (BMPR1a)²: another Example of a Single-Chain ReceptorTrap within the TGF-β Family

Another example of a TGF-β family member trap is (BMPR1a)², shownschematically in FIG. 2A. The (BMPR1a)² trap was compared with monomericBMPR1 a for neutralization of BMP2 (FIG. 14). The bivalent (BMPR1a)²trap was clearly able to neutralize BMP2 whereas monomeric BMPR1a showedpoor neutralization.

The multivalent polypeptide ligand binding agents described herein allowfor high affinity and specificity by single-chain multivalency. Thissingle-chain attribute is fundamentally different from existingmulti-chain agents such as Fc-based fusions (covalent dimer),E/K-coiled-coil-based fusions (non-covalent dimer), or describedcytokines and ligand traps that include fused multimerizing moieties.The present design can facilitate tissue penetration, thereby increasingaccess to sites of interest. The present design can also provide ashorter half life in systemic circulation, which can be desirable forcertain applications such as imaging and other diagnostic applications,as well as where ongoing abundant systemic distribution of theantagonist is not desirable. In addition, the present design permitslinkage of other cargo molecules (for example imaging agents likefluorescent molecules), toxins, etc.

Linkers can be designed to facilitate purification of the linker and/orligand binding agent. The exact purification scheme chosen willdetermine what modifications are needed, for example, additions ofpurification “tags” such as His tags is contemplated.

The general Structure I

(<bd1>-linker1)_(k)-[{<bd1>-(linker2-<bd2>)-Iinker3_(f)-}_(n)-(<bd3>)_(m)-(linker4-<bd4>)_(d)]_(h)

can be modified to add one or more cargo and/or accessory molecules(referred to collectively herein by R₁, R₂, R₃, R₄, etc.).

For example, to provide Structure V:

Where bd1, bd2, bd3, bd4, linker1, linker2, linker3, linker4, k, f, n,m, d, and h are defined as in Structure I.

Without limiting the generality of R substituients available, R₁, R₂,R₃, R₄, R₅, R₆, R₇, R₈, R₉, may be the same or different, may not bepresent and when present, may independently be one or more of:

-   -   A fusion protein for targeting such as an antibody fragment        (e.g. single chain Fv) and/or a single domain antibody (sdAb).    -   A radiotherapy and/or imaging agent such as a radionuceotide        (e.g. ¹²³I, ¹¹¹In, ¹⁸F, ⁶⁴C, ⁶⁸Y, ¹²⁴I, ¹³¹I , ⁹⁰Y , ¹⁷⁷Lu,        ⁶⁷Cu, ²¹³Bi, ²¹¹At) a florescent dye (e.g., Alexa Fluor, Cy dye)        and/or a fluorescent protein tag (e.g. GFP, DsRed).    -   A cytotoxic agent for chemotherapy such as doxorubicin,        calicheamicin, a maytansinoid derivatives (e.g. DM1, DM4), a        toxin (eg. truncated Pseudomonas endotoxin A, diphteria toxin).    -   A nano particle-based carrier such as polyethylene glycol (PEG),        a polymer-conjugated to drug, nanocarrier or imaging agent (e.g.        of a polymer N-(2-hydorxylpropyl)methacrylamide (HPMA), glutamic        acid, PEG, dextran).    -   A drug (e.g. doxorubicin, camptothecin, paclitaxel, palatinate).    -   A nanocarrier such as a nanoshell or liposome.    -   An imaging agent such as Supermagnetic Iron Oxide (SPIO)    -   A dendrimer    -   A solid support for use in ligand purification, concentration or        sequestration (e.g. nanoparticles, inert resins, suitable silica        supports).

In general, it will not be preferable to have cargo or accessorymolecules in all possible positions, as this may cause steric orelectrostatic complications. However, the effects of adding a cargo oraccessory molecule to any given position or positions on the structurecan be determined routinely in light of the disclosure herein bymodeling the linker between the binding domains and carrying outmolecular dynamics simulations to substantially minimize molecularmechanics energy and reduce steric and electrostatic incompatibilitybetween the linker and the member of the TGF-β superfamily as taughtherein.

It will frequently be preferable to add the cargo or accessory moleculeto the linker portion of the agent, rather to the binding domain, toreduce the likelihood of interference in binding function. However,addition to the binding domain is possible and could be desirable insome instances and the effect of such an addition can be determinedroutinely in advance by modeling the binding agent and the linker withthe proposed addition as described herein.

In certain embodiments of conjugation to cargo molecules and accessorymolecules, the following structures will be produced:

R-[bd]-(linker-[bd])_(n)

[bd]-(R-linker-[bd])_(n)

R-[bd]-(linker-[bd]-R)_(n)

R-[bd]-(R-linker[bd])_(n)

[bd]-(R-linker-[bd]-R)_(n)

R-[bd]-(R-linker-[bd]-R)_(n)

Conjugation methodologies are somewhat diverse but typically can beperformed using commercial kits that enable conjugation via commonreactive groups such as primary amines, succinimidyl (NHS) esters andsulfhydral-reactive groups. Some examples are; Alexa Fluor 488 proteinlabeling kit (Molecular Probes, Invitrogen detection technologies) andPEGylation kits (Pierce Biotechnology Inc.).

Many embodiments of the binding agents taught herein will have a lowermolecular mass, as compared with competing multivalent receptor-basedneutralizing agents.

In an embodiment of the invention there is provided ligand bindingagents wherein the intervening linker sequence, between theligand-binding domains, is composed of native amino acids, the sequenceof which is based on the receptor ectodomains (e.g. the various linkersshown in FIG. 2B and the “repeat” and “delete” linkers shown in FIG. 2D)or conservative substitutions of natural or unnatural amino acids intosuch regions or reversal of such natural or modified sequences. It willfrequently be considered preferable to use unstructured regions fromthese receptor ectodomains as the template for linker design. Oncelinkers have been designed, it will generally be preferred to test theireffectiveness using the procedures described herein or othersubstantially functionally equivalent procedures. Routine testing forimmunogenicity may be desired for in vivo use.

In some instances, it will be desirable to subject the polypeptide-basedlinking design of the ligand binding agents disclosed herein tooptimization of characteristics desired for a particular application.For example, the linker may be modified in length and composition basedon atomic-level simulations and knowledge-based design in order toimprove binding affinity, specificity, immunogenicity and stability.This is applicable to a wide range of molecular systems exhibitinghomomeric, heteromeric, dimeric and multimeric ligand-receptorstructural characteristics

Additional different binding domains can be incorporated to generatemultivalent traps with even higher binding potency.

In an embodiment of the invention, a non-naturally occurringsingle-chain hetero-bivalent polypeptide is produced by the inlinefusion of two or more different structured ligand-binding domains(denoted <bd1>, <bd2>, <bd3> and <bd4>) from the extracellular portionof distinct natural receptors, and which is not fused to any dimerizingor multimerizing moieties. In some instances, this polypeptide will havethe general structure <bd1>-linker2-<bd2>. In some instances, thebinding domains will be selected from the ectodomains of the TβR-II andTβRI receptors, and fused to produce hetero-bivalent single-chain trapsactive against TGF-β isoforms. In other instances, the binding domainswill be selected from the ectodomains of the ActR-IIa and BMPR-Iareceptors and fused to generate single-chain hetero-bivalent trapsactive against activin, myostatin and BMP isoforms. In otherembodiments, the binding domains are selected from other receptorstomembers of the TGF-β superfamily.

In another embodiment of the invention a non-naturally occurringsingle-chain hetero-trivalent polypeptide is produced by the inlinefusion of two or more different structured ligand-binding domains(denoted bd1 and bd2) from the extracellular portion of distinct naturalreceptors, and which is not fused to any dimerizing or multimerizingmoieties. In some instances, this polypeptide will have the generalstructure [bd1]-linker1-[bd2]-linker2-[bd2]. In other instances, thispolypeptide will have the general structure[bd1]-linker1-[bd1]-linker2-[bd2]. In some instances, [bd1] and [bd2]will be selected ectodomains of the TβR-II and TβRI receptors, and fusedto produce hetero-bivalent single-chain traps active against TGF-βisoforms. In other instances, bd1 and bd2 will be selected from theectodomains of the ActR-IIa and BMPR-Ia receptors and fused to generatesingle-chain hetero-bivalent traps active against activin, myostatin andBMP isoforms.

In another embodiment of the invention a non-naturally occurringsingle-chain hetero-tetravalent polypeptide is produced by the inlinefusion of two or more identical or different structured ligand-bindingdomains from the extracellular portion of natural receptors repeatedtwice or more times in various orders. In an embodiment to the inventionthis hetero-tetravalent polypeptide is not fused to any dimerizing ormultimerizing moieties. In one embodiment, this polypeptide will havethe general structure [bd1]-linker1-[bd2]-linker2-[bd1]-linker1-[bd2].In other instances, this polypeptide will have the general structure[bd1]-linker1-[bd1]-linker2-[bd2]-linker3-[bd2]. In one embodiment, thispolypeptide will have the general structure[bd1]-linker1-[bd2]-linker2-[bd2]-linker3-[bd1]. In some instances,[bd1] and [bd2] will be selected from the ectodomains of the TβR-II andTβR-I receptors, and fused to produce single-chain hetero-tetravalenttraps active against TGF-β isoforms. In other instances, [bd1] and [bd2]will be selected from the ectodomains of the ActR-IIa and BMPR-Iareceptors and fused to generate single-chain hetero-tetravalent trapsactive against activin, myostatin and BMP isoforms.

Specific non-limiting examples of embodiments of heteromericsingle-chain traps against TGF-β are represented schematically as wellas with full sequence details in FIGS. 15A and 15B.

A nucleotide sequence encoding a single-chain protein produced accordingto the teachings herein can be cloned and inserted into any suitablevector and therefore is very amenable to production (i.e. there is norequirement for two vectors, or one vector with two promoters, toexpress two receptor ectodomains).

The linker region provides a segment that is distinct from thestructured ligand binding domains and thus can be used for conjugationto accessory molecules (for example, molecules useful in increasingstability such as PEGylation moieties) or cargo molecules such ascontrast agents (for imaging) without having to chemically modify thebinding domains.

In an embodiment of the invention in which the ligand-binding domainsand the linker contain primarily natural sequences they would notordinarily be expected to be severely immunogenic or toxic in a typicalpatient.

Smaller size (for example, 50-60 kDa for (TβRII)² compared to 100-120kDa for TβRII-Fc or 150 kDa for monoclonal antibodies) will generally beexpected to increase access to target tissues.

Large scale production is an attainable goal. One 500 ml scale-up ofN-His (TβRII)² in 293 cells yielded 7 mg of purified protein.

In some instances, it may be desirable to permit a computer or othermachine capable of calculation to determine linker length according tothe disclosure herein. Thus, in an embodiment of the invention there isprovided a data storage medium comprising instructions for determiningthe minimum linker length. In an embodiment of the invention there isprovided a data storage medium comprising a means for identifyingacceptable minimal linker length.

Linker length will be considered acceptable when it permits binding ofbinding domains located on each of the N- and C-termini of the linker tobind their natural binding sites on their natural ligand such that, withboth binding domains so bound, the ligand is bound with a higheraffinity than it would be bound by binding of only one of the bindingdomains.

Methods Construction and Cloning of TGFβ Family Traps

1) Prototype (TβRII)²

Step 1: The mammalian expression vector pTT2-RIIE (De Crescenzo el al.,2003, J. Mol. Biol. 328: 1173), which contains a myc-tagged ectodomainof the human type II TGF-β receptor (TβRII) was cut with NotI and BamHIto eliminate E-coil/His regions.

Step 2: A second ectodomain of TβRII was PCR amplified from plasmidhuTGFβRII/pCDNA3 as template and using primers R2ECD3′Bamrev2 and R2ECD5′Not to incorporate a 3′ 6-His tag+Bam HI site, and a 5′ Not Irestriction site, respectively.

R2ECD3′Bamrev2: SEQ ID NO 1GACAGGATCCTAGTGATGATGGTGGTGATGGTCAGGATTGCTGGTGTTAT ATTC R2ECD 5′Not: SEQID NO 2 CACGGCGGCCGCCACGTTCAGAAGTCGGTTAATAAC

This PCR product was ligated to pCDNA3 cut with NotI and BamHI. Theinsert was verified by sequencing, re-excised by Not I/Bam HI digestionand then cloned into the vector from step 1, resulting in the assemblyof two TβRII ectodomains in tandem. The sequence of this construct wasverified by sequencing and the amino acid sequence of the prototype(TβRII)² protein is shown below and is presented schematically in FIG.6.

The gray sequence within brackets denotes the huTβRII signal peptidewhich is cleaved upon processing in 293 cells. The boxed sequences (EQK. . . LL and HH . . . HH), are Myc and His tags, respectively.

2) Modified N-His (TβRII)² and N-His TβRII Monomer

Step 1: Reversion of non-native amino acids GGR to native amino acidsIPP in the linkers of prototype (TβRII)²

The Not1 site within the linker coding sequence of prototype (TβRII)²created a GGR sequence (underlined in the amino acid sequences shownabove). This was reverted back to the native IPP sequence by PCR-basedmutagenesis. Internal primers 2XR2mutfor and 2Xmutrev span the region tobe mutated and two flanking primers pTT2 5′ and pTT2 3′ contain theflanking regions.

2XR2mutfor: CCTGACATCCCACCGCAGGTTCAGAAG SEQ ID NO 4 2Xmutrev:GAACGTGCGGTGGGATGTCAGGATTGC SEQ ID NO 5 pTT2 5′ ATACACTTGAGTGACAATGACASEQ ID NO 6 pTT2 3′ AAAATTCCAACACACTACTTTGCAATCT SEQ ID NO 7

The template used was pTT2-prototype(TβRII)² . Primers pTT2 5′ and2XR2mutrev were paired to create PCR fragment 1. Primers 2XR2mutfor andpTT2 3′ were paired to create PCR fragment 2. The PCR fragments 1 and 2were heated at 95° C. and allowed to anneal together. The flankingprimers were then used to amplify the assembled fragment 1+2. Theamplified fragment 1+2 was then cut with HindIII and BamHI and insertedinto pTT vector also cut with HindIII and BamHI. The resultant plasmidwas designated pTT2-native(TβRII)² .

Step 2: Elimination of C-terminal His and N-terminal Myc tags and fusionwith N-terminal His tag/thrombin cleavage site.

Two primers were designed, incorporating the desired sequence change(restriction sites, thrombin cleavage site, and eliminating the mycMyctag, and the His tag).

BamHI-Thr-IPP-R2ECD_for: GGATCCTTCAACCCGCGTATTCCGCCGCACGTTCAGA SEQ ID NO8 AGTCGGTT BstBI stop R2ECD rev: GCGTTCGAACTAGTCAGGATTGCTGGTGTTATATTCSEQ ID NO 9

These primers were used to generate two fragments by PCR usingpTT2-native(TβRII)² as a template: fragment 1XECD (monomer) and 2XECD(dimer). Both fragments were digested with BstBI and BamHI and clonedseparately into plasmid vector pTTVH8G (unpublished, derived from pTTvector; Durocher et al., 2002, Nucl. Acid. Res. 30: No. 2 e9) which hasthe human VEGF signal sequence/10 N-terminal amino acids of VEGF and8Xhis tag. The protein sequences of the resulting constructs are asfollows:

The gray sequence within brackets denotes the VEGF signal peptide. Theboxed sequences; (HH . . . HH and IPP), are the His tag and reverted IPPsequence, respectively.

3) N-His (TβRIIb)²

Step 1: Assembly of (TβRIIb)2 gene.

a. Plasmid pTT2-native(TβRII)² was cut with NotI and BamHI to eliminatethe second TβRIIECD.

b. A PCR fragment was generated using plasmid pRC/CMV-huTβRIIb(containing the human TβRIIb gene) as a template with the followingprimers:

R2ECD-3′Bamrev-2: GACAGGATCCTAGTGATGATGGTGGTGATGGT SEQ ID NO 12CAGGATTGCTGGTGTTATATTC and R2bECD 5′Not for:CACGGCGGCCGCCACGTTCAGAAGTCGGATGTGG SEQ ID NO 13

The resulting fragment comprised the TβIIb with a 3′6-His tag , Barn HIsite and stop codon, and Not I at the 5′ end. This fragment was cut withNot I and Barn HI and then cloned into the the vector from step a.

Step 2:

Using the plasmid from step 1b as template a PCR fragment was generated(that eliminates the N-terminal Myc tag and C-terminal His tags) withthe following primers:

BamHI-Thr-IPP-R2-ECD-for: GGATCCTTCAACCCGCGTATTCCGCCGCACGTTC SEQ ID NO14 AGAAGTCGGTT BstBI stop R2ECD rev: GCGTTCGAACTAGTCAGGATTGCTGGTGTTATATSEQ ID NO 15 TC

The resulting PCR fragment was digested with the appropriate enzymes andsubcloned into pTTVH8G. The protein sequence of the this trap is asfollows:

The gray sequence within brackets denotes the VEGF signal peptide. Theboxed sequence is the His tag.

4) (ActRIIB)² and ActRIIB Monomer

For construction of (ActRIIB)², three primer pairs (1+2, 3+4, 5+6) wereused to generate 3 PCR fragments (A, B and C) using a plasmid containingthe human ActRIIB sequence as a template.

Primer1: cgcagatctgcggccgcATGACGGCGCCCTGGGTGG SEQ ID NO 17CCCTCGCCCTCCTCTGGGGATCGCTGTGCGCA GGATCAGGATCAGAACAGAAGCTGATCaGCGPrimer2: CCGtGATCAGCTTCTGTTCTGATCCTGATCCTGCGCA SEQ ID NO 18CAGCGATCCCCAGAGGAGGGCGAGGGCCACCCA GGGCGCCGTCATgcggccgcagatctggc Primer3:GGCaGATCTCCGAGGAAGATTTACTAGGGCGTGGG SEQ ID NO 19 GAGGCTGAGACACGGGAGTGCATC Primer4: ccgactagtGGGGGCTGTCGGGGGTGGCTC SEQ ID NO 20 Primer5:ccgactagtGGGCGTGGGGAGGCTGAGAC SEQ ID NO 21 Primer6:cgctggatccCTAATGGTGATGATGGTGATGGG SEQ ID NO 22 TGGGGGCTGTCGGGGGTGGC

PCR Fragment A containins 5′ BgI II and NotI sites, the ATG start codon,the signal peptide and the 5′ half of the Myc tag and BcI I site. PCRfragment B contains the first ECD, with a BgIII site and the 3′ half ofthe Myc tag and SpeI site . Fragment C contains the second ECD, SpeIsite at the 5′ end, and a BamHI site, stop codon, the 6Xhis tag at the3′ end. These fragments were subcloned into pGemT vector (Promega),digested with the appropriate enzymes, and ligated together. Theresulting A+B+C fragment, which encodes the (ActRIIB)² single chaindimer, was cut using NotI and BamHI and inserted into pTT expressionvector. The ActRIIB monomer was assembled in a similar manner usingprimer pairs 1+2 and 3+6. The resulting constructs have the followingprotein sequences:

The gray sequence within brackets denotes the human ActRIIB signalpeptide. The boxed sequences; (EQK . . . LL and HH . . . HH), are Mycand His tags, respectively.

5) (BMPR1a)² and BMPR1a Monomer

For construction of (BMPR1a)², 2 primer pairs (1+2, 5+6) were used togenerate two PCR fragments (A, B) using a plasmid containing the humanBMPR1a (ALK3) sequence as a template.

Primer1for: SEQ ID NO 25 GCG AAG CTT ATG CCT CAG CTA TAC ATT TAC ATCPrimer4rev: SEQ ID NO 26 CGGC CTC CGG ATG CTG CTG CCA TCA AAA AAC GGPrimer5for: SEQ ID NO 27 CCGCG CGC CGG CAG AAT CTG GAT AGT ATG CTT CPrimer6rev: SEQ ID NO 28 CGAC AGG ATC CTA GTG ATG ATG GTG GTG ATG TCGAAT GCT GCC ATC AAA AAA CGG

PCR fragment A contains a 5′Hind III site, start codon, signal peptideand first

BMPR1aECD. PCR fragment B contains the second BMPR1aECD, 6Xhis tag, stopcodon and BamHI site.

These fragments were subcloned into pGemT vector (Promega), digestedwith the appropriate enzymes, and ligated together. The resulting A+Bfragment, which encodes the (BMPR1a)² single-chain dimer, was cut withHindIII and BamH1 and inserted into pTT2 expression vector. The BMPR1amonomer was assembled using primer pairs 1+6. The resulting constructshave the following protein sequences:

The gray sequence within brackets denotes the human BMPR1 a signalpeptide. The boxed sequence is the His tag.

Expression and Purification of Ligand Binding Agents

Modified human embryonic kidney cells (293-EBNA1 clone 6E) stablyexpressing EBNA1 were transfected using 25 kDa linear polyethylenimine(PEI) (Poysciences, Warrington, Pa.) as described below (and Durocher etal., 2002, Nucl. Acid Res. 30: e9)). The cells growing as suspensioncultures in Freestyle medium (Invitrogen) were transfected at 1×10⁶cells/ml with variable amounts of pTT vector plasmid DNA (for smallscale cultures), or a fixed amount of plasmid DNA (for large scaleculture), and 2 ug/ml PEI.

1. Small-Scale Transient Transfections:

Five hundred microliters of the suspension culture was distributed perwell in a 12-well plate. DNA was diluted in Freestyle medium (in avolume equivalent to one-tenth of the culture to be transfected), PEIwas added, and the mixture immediately vortexed and incubated for 10 minat room temperature prior to its addition to the cells. Following 3 hincubation with DNA—PEI complexes, culture medium was completed to 1 ml.The culture was harvested 5 days after transfection and the media wasclarified by centrifugation at 3500 g for 10 min and sterile filtered.Aliquots of conditioned media were analyzed for TGF-β binding activityvia SPR analyses (see below and FIG. 7A)

2. Large-Scale Cultures and Protein Purification:

Large scale cultures were processed as per (Pham et al., 2005:Biotechnol. Bioeng. 90: 332). Bioreactors of 1L (Biostat Q, B. Braun,Germany) were equipped with 45° pitched blade impellers and stirringspeed was maintained at 100 rpm. Surface aeration was applied with a gasmixture of nitrogen, carbon dioxide and oxygen at a gas-flow rate of 100standard cubic cm/min). The dissolved oxygen tension was controlled at40% air saturation. The temperature was maintained at 37° C. and the pHwas maintained at 7.15 with CO₂ at the beginning of the run and withNaHCO₃ (7.5% w/v) during the cell growth phase. A feed with 0.5% (w/v)TN1 peptone (OrganoTechnie) was done 24 hours post- transfection. Theculture medium was harvested 120 hours post transfection and trapprotein was purified by immobilized metal affinity chromatography onFractogel-Cobalt column as previously described (Cass et al., 2005,Protein Expr. Purl. 40: 77) except that wash and elution steps contained25 mM and 300 mM imidazole respectively. A 10 ml column packed with 5 cmTalon Metal Affinity Resin (BD Biosciences, Mississauga, Ont.) and wasequilibrated with 10 column bed volumes (CVs) of Talon Wash Buffer (TWB:50 mM sodium phosphate, 300 mM NaCl, pH 7). The conditioned medium waspassed through a 0.22 μm filter, and then loaded by gravity. The columnwas washed with 10 CVs of TWB and (TβRII)² was eluted in 1 ml fractionsusing 300 mM imidazole in TWB. Eluted trap protein was then desalted inPBS using a HiPrep 26/10 desalting column (GE-Healthcare) as recommendedby the manufacturer. Protein concentration was determined by Bradfordusing BSA as a standard. The progress of the various stages ofpurifcation for N-His (TβRII)² is seen in FIG. 8.

Surface Plasmon Resonance (SPR) Experiments

Analyses of Conditioned Media of Transfected 293-EBNA1 clone 6E cellsfor TGFβ-Binding Activity

Conditioned media from cells, transfected with increasing amounts ofpTT2-prototype (TβRII)² plasmid (to generate increasing percentages oftransfected cells ranging form 1-95%), were collected 5 dayspost-transfection and sterile filtered (0.22 μm). The samples werediluted to 1:100 or 1:20 using HBS buffer (10 mM HEPES, 150 mM NaCl, 3.4mM EDTA, and 0.02% Tween 20) prior to surface plasmon resonance (SPR)analysis of the (TβRII)² interaction with TGF-β3. SPR data was generatedusing a Biacore 3000 instrument (G.E. Healthcare Inc.) at 25° C. usingHBS as running buffer. Ligand was prepared by covalently immobilizing2000 resonance units (RUs) of TGF-β3, along with a mock blank controlsurface, onto a Biacore CM-5 sensor chip using standard amine couplingmethods. Samples were injected simultaneously over theTGF-β3-immobilized and blank surfaces for 240 s followed by a 240 sdissociation time at a flow rate of 10 μl/min. Specific (TβRII)²-TGF-β3interaction sensograms were generated by subtracting the sensogramgenerated from the blank surface from the one generated from theTGF-β3-immobilized surface. The sensograms were aligned to the injectionstart points using BiaEval software version 4.1 (Biacore Inc.), as shownin FIG. 7A.

Association and Dissociation of TβRII, Prototype (TβRII)² and TβRII-Fcwith TGF

Sensograms comparing (TβRII)² with TβRII-Fc and E. coli-producedmonomeric TβRII ectodomain were generated. The ligand surface andinjection conditions were the same as described above except injectiontimes were for 120 s. Solutions containing 200 nM purified TβRII and 25nM TβRII-Fc in HBS buffer were used for analysis. (TβRII)²-conditionedmedia (from 95% transfected cells) was diluted 1:20 in HBS buffer. TheSPR sensograms generated from these sample injections were aligned totheir injection start point and normalized to a maximum RU response of100 using Biacore BiaEval software version 4.1 (Biacore Inc.), as shownin FIG. 7B.

Comparison of the TGFβ-Binding Efficacies of Purified (TβRII)², TβRII-Feand TβRII Monomer

Solutions containing a TβRII variant ((TβRII)², TβRII-Fc or E.coli-produced TβRII) and 5 nM of TGF-β (β1 or β3) were pre-incubated andthen injected over covalently immobilized 1 D11 anti-TGF-β antibody (R&DSystems) under mass-transport limiting conditions to measure free TGF-β.SPR data was generated using a Biacore 3000 instrument (G.E. HealthcareInc.) using HBS as running buffer. A high density 1 D11 surface(approximately 10,000 RUs) and matching blank control surface werecreated on a Biacore CM-5 sensorchip using standard amine couplingmethods. A twenty-fold stock concentration of TGF-β (100 nM in 10 mMacetic acid) was used. This gave the final 1-fold assay concentration ofTGF-β (5 nM) when 10 μl was added to 190 μl HBS containing a TβRIIvariant at a 1.05 times final concentration. Blank injection sampleswere made from 10 μl 10 mM acetic acid mixed with 190 uL HBS. TGF-β wasadded to the TβRII variant solution using the TRANSFER command, mixed,and incubated for 120 s at 4° C. prior to injection over the 1D11surface. Using the KINJECT command, samples were simultaneously injectedfor 5 min over the 1D11 and control surfaces with a 30 s dissociationtime at a flow rate of 5 μl/min at 25° C. The 1 D11 surface wasregenerated for the next cycle by injecting 10 mM HCl for 15 s at 20μl/min using the INJECT command. All sensogram analysis was carried outusing Biacore BiaEvaluation software v4.1 (G.E. Healthcare Inc.). TβRIIvariant sensograms were aligned to the injection start point, anddouble-referenced using the control surface and blank injectionsensograms. The plateau levels (which measure the amount of free TGFβ)were taken from the average value of the stabilized dissociation phaseof each double-referenced sensogram. Examples are shown in FIGS. 9B, 10Cand 12B.

Comparison of the Antagonistic/Inhibitor Potencies of Various BindingAgents by Luciferase Reporter Assays

1. Luciferase Assay for a TGF-β binding agent in mink lung epithelial(Mv1Lu) cells.

Mink lung epithelial cells, stably transfected with the TGF-β-responsivePAI-1 promoter fused to the firefly luciferase reporter gene (Abe etal., 1994, Anal. Biochem. 216: 276), were used. These cells were platedin 96-well tissue culture plates (2×10⁴ cells/well) in Dulbecco'smodified Eagle's medium containing 5% fetal bovine serum and wereallowed to attach for at least 6 h at 37° C. Cells were then washed withphosphate buffered saline (PBS), and the medium was replaced byDulbecco's modified Eagle's medium containing 1.0% fetal bovine serumand 0.1% bovine serum albumin (DMEM-1, 0.1% BSA). Various concentrationsof purified (TβRII)² or (TβRIIb)² trap or TβRII-Fc (either from R&DSystems or collaborator Dr. Herbert Lin) were mixed with 20 pM TGF-β1 inDMEM-1, 0.1% BSA) and added to the cells. After 16 hr. incubation at 37°C., the medium was removed, and the cells were washed once with PBS.Cells were then lysed with 25 μl reporter lysis buffer (Promega Corp.)and assayed for luciferase activity using the Promega luciferase assaykit according to the manufacturer's instructions. Luminescence wasmeasured in a MRX (Dynex Inc.) or Lumioskan RS (Global MedicalInstrumentation, Inc.) microplate reader. The activity is expressed asthe percentage of the maximum TGF-β1 activity (i.e. in the absence ofany antagonist) or relative luciferase units (RLU) (see examples shownin FIGS. 9A, 10D and 12A).

2. Luciferase assay for ActRIIB binding agents in A204 cells.

A204 cells (rhabdomyosarcoma, ATCC) were plated in 48-well cultureplates (5×10⁴ cells/well) in McCoy's 5A Media (ATCC) supplemented with10% Fetal bovine serum. After 24 hrs. the cells were transfected with(CAGA)₁₂MLP-Luc (luciferase reporter responsive to Activin andmyostatin; Dennler et al., 1998, EMBO J. 17: 3091) and pRL-CMV(constitutive renilla reporter for normalization of transfections,Promega Corp.) using Lipofectamine 2000 transfection reagent, accordingto the manufacturer's specifications (Gibco-BRL). After 24 hours thecells were washed once with DMEM-1, 0.1% BSA and then treated with 4 nMhuman myostatin (GDF8, R&D Systems) without or with increasingconcentrations of ActRIIb trap or ActRIIb-Fc (R&D Systems) for 6 hrs,37° C. The cells were then washed once with PBS and lysed with 50 μl 1×Passive lysis buffer. The lysates were measured by a dualfirefly/renilla luciferase reporter kit, according to the manufacturer's(Promega Corp). The activity is expressed as firefly RLU normalized torenilla (see example shown in FIG. 13).

3. Luciferase assay for BMPR1a binding agents in C2C12BRA cells.

C2C12BRA cells (mouse myoblast cells stably transfected with aBMP-luciferase reporter; Zilderberg et al., 2007, BMC Cell Biology 8:41) were plated onto 96-well culture plates (5×10³ cells/well) in DMEMsupplemented with 10% fetal bovine serum. After 24 hrs. the cells werewashed once with DMEM-1, 0.1% BSA and then treated with 1 nM human BMP2with or without increasing amounts of BMPR1a trap or BMPR1a-Fc (R&DSystems) for 24 hrs. at 37° C. The cells were then washed once with PBSand lysed with 50 μl 1× Reporter lysis buffer. The lysates were measuredby a firefly luciferase reporter kit, according to the manufacturer's(Promega Corp). The activity is expressed as firefly RLU (see exampleFIG. 14).

Neutralization/Inhibition of TGF-β-Induced 4T1 Cell Invasion by TGFβBinding Agents

4T1 cells (mouse mammary carcinoma, ATCC) were seeded onto BD BioCoatMatrigel invasion chambers (BD Biosciences) at 1×10⁵ cells/chamber inDMEM containing no serum, without or with 100 pM TGF-β, and with 400 nMprototype (TβRII)² trap or TβRII-Fc. The cells were allowed to invadethrough the matrigel into the bottom chamber for 18 hrs at 37° C. Thecells on the upper side of matrigel membrane were removed by scrapingand invaded cells were stained/fixed with 0.2% crystal violet, 100%ethanol. The number of invaded cells was quantified for 4 fields ofequal size via light microscopy. The example shown in FIG. 9C shows theaverage % invasion (relative to +TGF-β control) from 3 experiments.

Western Blot to Determine N-His (TβRII)² Protein Stability in Serum

Equal amounts N-His (TβRII)² protein were incubated for 1-7 days at 37°C. in DMEM+10% fetal bovine serum. Equal aliquots were electophoresed ina 8% SDS-reducing gel followed by western blotting and probing withanti-TβRII antibody (R&D Systems). The result is shown in FIG. 11A.

TABLE 1 Linker characteristics for select examples of single-chain trapsof TGF-β-family growth factors. Reference Linear Minimum Receptorstructures Residues distance residues Single- Targeted ectodomain (PDBin “natural” (Å) for required chain trap ligand(s) used entries) linkerlinkage for linkage (ActR-IIa)² BMP-7 ActR-IIa-ED 2GOO, 28 70 28 1LX5(ActR-IIb)² Activin ActR-IIb-ED 1S4Y, 25 45, 50 18 Myostatin 1NYU(BMPR-Ia)² BMP-2 BMPR-Ia-ED 2GOO, 41 60 24 1ES7 (TβR-II)² TGF-β1TβR-II-ED 1KTZ, 35 80 32 TGF-β3 1PLO, 1M9Z (TβR-IIb)² TGF-β1 TβR-IIb-ED1KTZ, 60 80 32 TGF-β3 1PLO, 1M9Z Minimum number of residues required forlinkage represents the structure-based linear distance for linkage (Å)divided by a factor of 2.5.

TABLE II In addition to linkers disclosed elsewhere herein,the followingpolypeptide sequences may be useful as linkers or components thereof.These polypeptides may be useful when produced using either L- orD-amino acids. However, with respect to SEQ ID NOs 82 to 118, use ofD-amino acids will frequently be preferred.COOH-IPPHVQKSVNNDMIVTDNNGAVKFP-NH2 SEQ ID NO 82 COOH-SEEYNTSNPD NH2 SEQID NO 83 COOH IPPHVQKSDVEMEAQKDEIICPSC SEQ ID 84NRTAHPLRHINNDMIVTDNNGAVKFP-NH2 COOH-SEEYNTSNPD NH2 SEQ ID NO 85COOH-AALLPGAT NH2 SEQ ID NO 86 COOH-PTTVKSSPGLGPVE NH2 SEQ ID NO 87COOH-AILGRSE NH2 SEQ ID NO 88 COOH-EMEVTQPTSNPVTPKPPYYNI NH2 SEQ ID NO89 COOH-SGRGEAET NH2 SEQ ID NO 90 COOH-EAGGPEVTYEPPPTAPT NH2 SEQ ID NO91 COOH-1QNLDSMLHGTGMKSDSDQKKSEN SEQ ID NO 92 GVTLAPED NH2COOH-PVVIGPFFDGSIR N SEQ ID NO 92 COOH-SEEYNTSNPDIPPHVQKSVNNDMIV SEQ IDNO 93 TDNNGAVKFP NH2 COOH-SEEYNTSNPDIPPHVQKSDVEMEAQKDEII SEQ ID NO 94CPSCNRTAHPLRHINNDMIVTDNNGAVKFP NH2 COOH-EAGGPEVTYEPPPTAPTSGRGEAET NH2SEQ ID NO 95 COOH-PVVIGPFFDGSIRQNLDSMLHGTGMKS SEQ ID NO 96DSDQKKSENGVTLAPED N COOH-PVVIGPFFDGSIRGNLDSMLHGTGMKSDSDQK SEQ ID NO 97KSENGVTLAPED NH2 COOH-SEEYNTSNPDGPPHVQKSVNNDMIVT SEQ ID NO 98 DNNGAVKFPNH2 COOH-EAGGPEVTGEPPPTAPTSGRGEAET NH2 SEQ ID NO 99COOH-SEEYNTSNPDGGRHVQKSDVEMEAQKDE SEQ ID NO 100IICPSCNRTAHPLRHINNDMIVTDNNGAVKFP NH2 COOH-SEEYNTSNPDGGPHVQKSVNNDM SEQ IDNO 101 IVTDNNGAVKFP NH2 COOH-SEEYNTSNPDGGRHVQKSVNND SEQ ID NO 102MIVTDNNGAV KFP N- COOH-SEEYNTSNPSGGGSGGGSGGGMEAQKDEI SEQ ID NO 103ICPSCNRTAHPLRHINNDMIVTDNNGAVKFP NH2 COOH-SEEYNTSNPSGGGSGGKSVNNDMIV SEQID NO 104 TDNNGAVKFP NH2 COOH-SEEYNTSNPSGGGSGGGSGGGDMIV SEQ ID NO 105TDNNGAVKFP NH2 -SEEYNTSNPDIPPHVQKSGGGSGGGSGGGSGGGS SEQ ID NO 106GGGSGGGSGGNNDMIVTDNNGAVKFP NH2 COOH-SEEYNTSNPDGGGSGGGSGGGSGGGSGGGSG SEQID NO 107 GGSGGGSGGGSGGNNDMIVTDNNGAVKFP NH2 COOH-SEEYNTSNPDIPPHVQKSVNNDMSEQ ID NO 108 IVTDNNGAVKFP NH2 COOH-SEEYNTSNPDIPPHVQKSVNNDM SEQ ID NO109 IPPHVQKSVNNDMIVTDNNGAVKFP NH2 COOH-SEEYNTSNPPHVQKSVNNDMIVT SEQ ID NO110 DNNGAVKFP NH2 COOH-SEEYNTSNPDGGGGGGGGIPPHVQK SEQ ID NO 111SVNNDMIVTDNNGAVKFP NH2 COOH-SEEYNTSNPDGGGSGGGSGGGSIPPH SEQ ID NO 112VQKSVNNDMIVTDNNGAVKFP NH2 COOH-SEEYNTSNPDIPPHVQKSDVEMEAQK SEQ ID NO 113DEIICPSCNRTAHPLRHINNDMIVTDNNG AVKFP NH2COOH-SEEYNTSNPDIPPHVQKSDVEMEAQKDE SEQ ID NO 114RTAHPLRHINNDMIVTDNNGAVKFP NH2 COOH-EAGGPEVTYEPPPTAPTSGRGEAET NH2 SEQ IDNO 115 COOH-EAGGPEVTYEPPPTAPTGGGGGGGGGGS SEQ ID NO 116 GRGEAET NH2COOH-PVVIGPFFDGSIRQNLDSMLHGTGMKSD SEQ ID NO 117 SDQKKSENGVTLAPED NH2COOH-PVVIGPDGSIRQNLDSHGTGMKSDSDQ SEQ ID NO 118 KKSENGVTLAPED NH2

Also contemplated are nucleic acid sequences encoding such linkers.

1. A multivalent binding agent with affinity for a member of the TGF-βsuperfamily, said agent comprising the general structure I:(<bd1>-linker1)_(k)-[{<bd1>-linker2-<bd2>-linker3_(f)-}_(n)-(<bd3>)_(m)-(linker4-<bd4>)_(d)]_(h),where: -n and h are independently greater than or equal to 1; -d, f, mand k are independently equal to or greater than zero; -bd1, bd2, bd3and bd4 are polypeptide binding domains having an affinity for the samemember of the TGF-β superfamily, with bd1, bd2, bd3, and bd4 beingindependently the same or different from each other; and, -linker1,linker2, linker3 and linker4 are unstructured polypeptide sequences;wherein the number of amino acids in each linker is determinedindependently and is greater than or equal to X/2.5; where, X equals theshortest linear distance between: (a) the C-terminus of an isolated formof the binding domain that is located at the N-terminus of the linkerand that is specifically bound to its ligand; and, (b) the N-terminus ofan isolated form of the binding domain that is located at the C-terminusof the linker and that is specifically bound to its ligand.
 2. The agentof claim 1 wherein the member of the TGF-β superfamily to which thebinding domains have affinity is selected from the group consisting of:TGF-β1, TGF-β2, TGF-β3, activin βA, activin βB, activin βC, activin βE,bone morphogenic protein (BMP) 2, BMP 3, BMP4, BMP 5, BMP 6, BMP 7, BMP8, BMP 9, BMP 10, BMP 11, BMP 12, BMP 13, BMP 14, BMP 15, growthdifferentiation factor (GDF) 1, GDF 3, GDF 8, GDF 9, GDF 15, Nodal,Inhibin α, anti-Mullerian Hormone, Lefty 1, Lefty 2, arteman, Persephinand Neurturin.
 3. The agent of claim 2 wherein the member of the TGF-βsuperfamily to which the binding domains have affinity is selected fromthe group consisting of: TGF-β1, TGF-β2, TGF-β3, BMP2, GDF 8, andactivin.
 4. The agent of any preceding claim wherein the linker isbetween 25 and 60 amino acids in length
 5. The agent of any precedingclaim wherein bd4 is the same as bd1, bd 2 is the same as bd3, h>0, andd, f, m, and n=1.
 6. The agent of any preceding claim comprising any oneor more of SEQ ID No 75 to
 82. 7. The agent of any preceding claimcomprising one or more of SEQ ID NO 31-42 or 49-74 as a linker sequence.8. The agent of any preceding claim wherein one or more of bd1, bd2,bd3, and bd4 is selected from one of SEQ ID NO 43-48.
 9. The agent ofany preceding claim having the general structure V:

Wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, may be the same ordifferent, may not be present and when present, may independently be oneor more of a fusion protein for targeting, a single domain antibody, aradiotherapy agent, an imaging agent, a fluourescent dye, a fluorescentprotein tag, a cytotoxic agent for chemotherapy, a nano particle-basedcarrier, a polymer-conjugated to drug, nanocarrier or imaging agent, astabilizing agent, a drug, a nanocarrier, a support, and a dendrimer.10. A method of designing a multivalent linker useful in modulatingresponsiveness of a cell to a member of the TGF-β superfamily, saidmethod comprising: a) identifying a member of the TGF-β superfamily ofinterest; b) obtaining two polypeptide binding domains having affinityfor different sites on the member of the TGF-β superfamily member; c)obtaining an unstructured polypeptide linker of at least a number ofamino acids equal to (X/2.5), wherein the number of amino acids in eachlinker is determined independently and is greater than or equal toX12.5; where, X equals the shortest linear distance between: (i) theC-terminus of an isolated form of the binding domain that is located atthe N-terminus of the linker and that is specifically bound to itsligand; and, (ii) the N-terminus of an isolated form of the bindingdomain that is located at the C-terminus of the linker and that isspecifically bound to its ligand; and d) modeling the linker having oneof the binding domains covalently attached at each end, and carrying outmolecular dynamics simulations to substantially minimize molecularmechanics energy and reduce steric and electrostatic incompatibilitybetween the linker and the member of the TGF-β superfamily.
 11. Themethod of claim 10 further including a step e) producing a fusionprotein comprising the two polypeptide binding domains joined by theunstructured polypeptide linker.
 12. An isolated polypeptide having atleast 80% sequence identity to a natural unstructured region in theextracellular portion of a receptor for a member of the TGF-βsuperfamily and being substantially free of structured regions capableof specific binding to a member of the TGF-β superfamily.
 13. Theisolated polypeptide of claim 12 having at least 80% sequence identityto one or more of SEQ ID NO 31-42 and SEQ ID NOs 49-74.
 14. Apolypeptide comprising a region having at least 80% sequence identity toone or more of SEQ ID NOs 53-74 and SEQ ID NOs 82-118.
 15. Thepolypeptide of claim 14 having a region with at least 90% sequenceidentity to one or more of SEQ ID NOs 53-74.
 16. A polypeptide havingbetween 43% and 99% sequence identity to a naturally unstructured regionin the ectodomain of a receptor for a member of the TGF-β superfamily.17. A nucleic acid sequence encoding a polypeptide of any precedingclaim.
 18. A method of modulating the response of a cell to a TGF-βsuperfamily member in its environment, said method comprising exposingthe cell to an agent of any one of claims 1 to
 9. 19. A data storagemedium comprising instructions for determining the minimum linker lengthin step c of claim
 10. 20. A data storage medium comprising a means foridentifying acceptable minimal linker length in step c of claim
 10. 21.Use of an agent of claim 1 to concentrate ligand in a sample.
 22. Use ofan agent of claim 1 to purify ligand.
 23. Use of an agent of claim 9 indiagnosis of a condition characterized in whole or part by anabnormality in levels of one or more TGF-β superfamily members in thebody or a portion thereof.
 24. Use of an agent of claim 9 in targetingdelivery of a compound to a site of interest within a body.